Miniprep/Kit-free high-throughput protocol-PDF

Background

This protocol is adapted from “Molecular Cloning: A Laboratory Manual”, Second Edition, Sambrook, Fritsch, and Maniatis. It is a quick, inexpensive way to purify large numbers of plasmids (I used to routinely do 80 at a time.–Kathleen) and yields DNA that is clean enough for sequencing or for use as a PCR template.

Protocol

1. Transfer 1.5 mL of an overnight culture containing your plasmid to an eppendorf tube and spin at 5000 rpm for 5 min in a tabletop centrifuge to pellet the cells.
2. Remove and discard the supernatent.
3. Add 300 μL STET buffer, and resuspend cells by vortexing.
4. Add 10 μL lysozyme (10 mg/mL), vortex, and submerse in boiling water for 40 sec.
5. Spin for 30 min in a tabletop centrifuge at maximum speed at 4 ˚C.
6. Remove pellet from each tube with a toothpick. The cellular debris should stick well to the toothpick. Try to insert and remove the toothpick from the center of the tube so you don’t get any cellular debris on the sides of the tube.
7. Add 300 μL ice cold isopropanol to precipitate the DNA (or 300μL of 2:1 isopropanol:ammonium acetate, mixed just before you use it. See this discussion of precipitating nucleic acids.)
8. Spin for 10 min in a tabletop centrifuge at maximum speed at 4 ˚C.
9. Remove supernatent.
10. Add 200 μL ice cold 80% ethanol to wash pellet and spin for 5 min in a tabletop centrifuge at maximum speed at 4 ˚C.
11. Remove supernatent.
12. Dry pellet (air dry at room temperature or 37 ˚C or dry in a speedvac).
13. Rehydrate in 50 μL TE.

Buffers

STET

• 8% sucrose
• 50 mM Tris-HCl, pH 8
• 0.5% Triton X-100
• 50 mM EDTA

TE

• 10 mM Tris-HCl, pH 8
• 1 mM EDTA